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1.
The single calmodulin gene ( CaM) of the green alga Mougeotia scalaris (Hassall) was cloned, sequenced and the CDNA inserted into the prokaryotic expression vector pGEX-2T. The recombinant calmodulin protein (CAM) was expressed as a fusion product together with glutathione S-transferase and isolated on glutathione sepharose. After cleavage and purification, the CaM was characterized by Ca 2+-dependent shift in SDS-PAGE, by activation of cyclic 3′,5′nucleotide phosphodiesterase (PDE) and sensitivity to the inhibitors trifluoperazine and calmidazolium, with native Mougeotia CaM as control. Using Ca 2+ buffers in the PDE test, affinity to Ca 2+ of Mougeotia CaM was found to be diminished fivefold compared to maize or bovine brain CaMs. There was also a 20-fold increase of half maximal activation (K act) in the PDE test for Mougeotia CaM relative to maize CaM, while the K act of maize CaM to that of bovine brain CaM was almost the same. The derived amino acid sequences of CaM from Mougeotia and Zea mays revealed three major conservative amino acid exchanges, including unique 105-Trp ( Mougeotia) → Leu (maize). In Mougeotia CaM the 105-Trp, including the neighbouring side chains of 92-Phe and 141-Phe, putatively form a hydrophobic ring interaction, as revealed by molecular modelling. 相似文献
2.
Cyclic AMP (cAMP) acts as a second messenger and is involved in the regulation of various physiological responses. Recently, we identified the cAMP-synthesis/hydrolysis enzyme CAPE, which contains the two catalytic domains adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) from the liverwort Marchantia polymorpha. Here we characterize the PDE domain of M. polymorpha CAPE (MpCAPE-PDE) using the purified protein expressed in E. coli. The Km and Vmax of MpCAPE-PDE were 30 µM and 5.8 nmol min?1 mg?1, respectively. Further, we investigated the effect of divalent cations on PDE activity and found that Ca2+ enhanced PDE activity, suggesting that Ca2+ may be involved in cAMP signaling through the regulation of PDE activity of CAPE. Among the PDE inhibitors tested, only dipyridamole moderately inhibited PDE activity by approximately 40% at high concentrations. Conversely, 3-isobutyl-1-methylxanthine (IBMX) did not inhibit PDE activity. 相似文献
3.
Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca 2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L‐type Ca 2+ channels raise questions on the extent of conservation of Ca 2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)‐sensitive Ca 2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L‐type (DHP‐sensitive) Ca 2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP‐specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8‐Br‐cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H‐89, an inhibitor of protein kinase‐A (PKA), reduced the current and inhibited the effect of 8‐Br‐cAMP. The data suggest modulation of L‐type Ca 2+ channels of Drosophila via a cAMP‐PKA mediated pathway. While there are differences in L‐type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP‐sensitive Ca 2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 491–500, 1999 相似文献
4.
Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM ( hCaM M124C– mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca 2+–CaM, Ca 2+–CaM–PDE1A, and Ca 2+–CaM–MLCK complexes, CPZ apparent dissociation constants ( Kds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca 2+– hCaM M124C– mBBr quenched the fluorescence ( Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca 2+– hCaM M124C– mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca 2+–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca 2+–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
5.
3′,5′-cAMP plays an important role as a second messenger molecule controlling multiple cellular processes in the brain. Its
levels are decreased by phosphodiesterases (PDEs), responsible for hydrolysis of intracellular cAMP. A part of the PDE activity
is dependent on the effect of calcium, mediated by its binding to calmodulin. During oxidative stress, precisely these changes
in calcium concentration are responsible for cell damage. We have examined the effects of oxidative stress conditions on the
activity of PDE in rat brain homogenates. We found a different influence of activated lipid peroxidation conditions (Fe 2+ with ascorbate and increased temperature) on the calcium-dependent and calcium-independent PDE activity. The inhibition of
Ca 2+-dependent PDE was observed, while Ca 2+-independent PDE was not influenced. We assume that it might be the impact of lipid peroxidation products or any mechanism
activated by the higher temperature on the interaction of the Ca 2+-dependent isoform of PDE with the complex calcium-calmodulin. Another explanation might be that the formation of the functioning
calcium-calmodulin complex is impossible in these conditions. 相似文献
6.
In vertebrate rods, dark and light conditions produce changes in guanosine 3′,5′‐cyclic monophosphate (cGMP) and calcium (Ca 2+) levels, which are regulated by the opposing function of several proteins. During the recovery of a bright flash, guanylate cyclase (GUCY) helps raise cGMP to levels that open cGMP‐gated calcium sodium channels (CNG) to increase Na + and Ca 2+ influx in the outer segment. In contrast, light activates cGMP phosphodiesterase 6 (PDE6) causing rapid hydrolysis of cGMP, CNG closure, and reduced Na + and Ca 2+ levels. In Pde6b mouse models of retinitis pigmentosa (RP), photoreceptor death is preceded by abnormally high cGMP and Ca 2+ levels, likely because of continued synthesis of cGMP by guanylate cyclases and unregulated influx of Ca 2+ to toxic levels through CNG channels. To reverse the effects of Pde6b loss of function, we employed an shRNA knockdown approach to reduce the expression of Gucy2e or Cnga1 in Pde6bH620Q photoreceptors prior to degeneration. Gucy2e‐ or Cnga1‐shRNA lentiviral‐mediated knockdown GUCY2E and CNGA1 expression increase visual function and photoreceptor survival in Pde6bH620Q mice. We demonstrated that effective knockdown of GUCY2E and CNGA1 expression to counteract loss of PDE6 function may develop into a valuable approach for treating some patients with RP. 相似文献
7.
Synaptic plasticity, neuronal activity‐dependent sustained alteration of the efficacy of synaptic transmission, underlies learning and memory. Activation of positive‐feedback signaling pathways by an increase in intracellular Ca 2+ concentration ([Ca 2+] i) has been implicated in synaptic plasticity. However, the mechanism that determines the [Ca 2+] i threshold for inducing synaptic plasticity is elusive. Here, we developed a kinetic simulation model of inhibitory synaptic plasticity in the cerebellum, and systematically analyzed the behavior of intricate molecular networks composed of protein kinases, phosphatases, etc. The simulation showed that Ca 2+/calmodulin‐dependent protein kinase II (CaMKII), which is essential for the induction of synaptic plasticity, was persistently activated or suppressed in response to different combinations of stimuli. The sustained CaMKII activation depended on synergistic actions of two positive‐feedback reactions, CaMKII autophosphorylation and CaMKII‐mediated inhibition of a CaM‐dependent phosphodiesterase, PDE1. The simulation predicted that PDE1‐mediated feedforward inhibition of CaMKII predominantly controls the Ca 2+ threshold, which was confirmed by electrophysiological experiments in primary cerebellar cultures. Thus, combined application of simulation and experiments revealed that the Ca 2+ threshold for the cerebellar inhibitory synaptic plasticity is primarily determined by PDE1. 相似文献
8.
Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M 2AChR pharmacological profile. Therefore, a novel Ca 2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58?kDa) being the 58?kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [ 3H]cGMP and [ 3H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg 2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC 50 of 4.9 and 4.6?μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M 2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed. 相似文献
9.
Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca 2+ signals. Previously, we reported divergent soybean ( Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions
of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca 2+-dependent electrophoretic mobility shift and Ca 2+ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies.
Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest
that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels. 相似文献
10.
Abstract Calmodulin (Cam), the heat-stable, ubiquitous, Ca 2+-dependent regulator protein, has been purified to apparent homogeneity from germinating radish seeds ( Raphanus sativus). The characteristics of radish Cam-molecular weight, absorption spectrum, Ca 2+-dependent activation of brain phosphodiesterase (PDE)-are very similar to those described for Cam from other plant materials. Radish Cam, like other plant Cam, shows some differences to Cam of calf brain. The total amount of Cam in radish embryos at 24 h of germination is ca. 37 μg g −1 fresh weight. Approximately 95% of the total amount of Cam is present in the soluble fraction (supernatant at 100,000 g). The level in the embryo axis strongly increases in the first 24 h of germination (+540%); this increase is strongly reduced when the germination is inhibited by abscisic acid (ABA). In the presence of Ca 2+, no ‘free’ Cam (i.e. not bound to other structures) is present in the soluble fraction, suggesting that, during early germination, Cam level is a limiting factor for the activities of Ca 2+ -Cam-dependent systems. These studies suggest that Cam plays an important role in the early phases of seed germination. An inhibitor of the Ca 2+-Cam-dependent phosphodiesterase is present in the soluble fraction from radish embryos; this substance decreases during germination. A possible role of this inhibitor during the early germination phases is hypothesized. 相似文献
11.
Inhibitors of phosphodiesterase type III (PDE III) enhance cardiac contractile force by elevating the intracellular calcium concentration [Ca 2+] i by impairing cAMP degradation thus increasing cAMP levels. The drugs are more effective in healthy than in failing hearts since basal cAMP production is diminished in the latter. However, long term treatment with PDE-III inhibitors does not appear to be beneficial due to increased risk of potentially lethal arrhythmias caused by augmentation of [Ca 2+] i[1). This risk should be absent in Ca 2+ sensitizers. Recently, thiadiazinone derivatives have been synthetized in which the potency for Ca 2+ sensitization is many-fold larger than the potency for PDE-III inhibition. The Ca 2+-sensitizing action resides in the [+]-enantiomers, while the [–]-enantiomers show weak PDE-III inhibition. In the enantiomer pair [+]-EMD 60263 and [–]-EMD 60264, only the former concentration-dependently increased force of contraction in isolated cardiac preparations and myocytes. In the Langendorff-perfused guinea-pig heart, force was reversibly increased, whereas [–]-EMD 60264 even produced a negative inotropic response despite of its PDE inhibitory activity. Heart rate, however, was reduced by both enantiomers. Perfusion pressure remained unaffected. The effects were fully reversible upon wash-out of the enantiomers. [+]-EMD 60263 also enhanced cell shortening of human myocytes from both normal and failing hearts. In contrast to the opposite effects on contractility, both enantiomers prolong the action potential duration by blocking the rapidly activating component of the delayed rectifier K + current. Thus they also possess class III antiarrhythmic activity. The therapeutic potential of these agents has yet to be assessed in clinical studies. 相似文献
12.
The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe 2(L) 2(NO) 4] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca 2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca 2+- ATPase, thus disturbing the balance of Ca 2+ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs. 相似文献
13.
It was established that microvessels of a bovine cortex exhibit significant cyclic 3,5-adenosine monophosphate phosphodiesterase (cAMP PDE) and cyclic 3,5-guanosine monophosphate phosphodiesterase (cGMP PDE) activities. These activities are dependent on the presence of Mg 2+. Absence of Ca 2+ was virtually without effect. When both Mg 2+ and Ca 2+ were absent, PDE activities increased compared with activities observed in the absence of Mg 2+. Xanthines (caffeine, theobromine, and theophylline) were better inhibitors of cAMP PDE than of cGMP PDE. Imidazole, in very high concentration (1×10 –2M) only, exhibited PDE stimulatory activity at high concentrations of both substrates. Otherwise, it exhibited PDE-inhibitory properties. 相似文献
14.
ATP-dependent Sr 2+ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination
mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr 2+ uptake and Ca 2+ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for
Ca 2+ and Sr 2+ uptake. The apparent K
m and V
max of ATP-dependent Sr 2+ uptake were both higher than those for Ca 2+ uptake. ATP-dependent Sr 2+ uptake by BLM vesicles diminished in the presence of 0.1 μ M Ca 2+ and was more markedly inhibited by 1 μM Ca 2+. Hill plots of Sr 2+ uptake data with and without 0.1 μ M Ca 2+ showed that the cooperative behavior of Sr 2+ uptake was not changed by Ca 2+. In the presence of 0.1 μ M Ca 2+, the affinity of the transport system for Sr 2+ and the velocity of Sr 2+ uptake in the BLM were both decreased. However, the rate of Ca 2+ uptake was not diminished by Sr 2+ concentrations of <1.6 μ M. These results suggest that Ca 2+ is preferentially transported in the renal cortex BLM when Ca 2+ and Sr 2+ are present at the same time. 相似文献
15.
The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca 2+ concentration ([Ca 2+] i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca 2+-sensitive fluorescence measurements. Increases in [Ca 2+] i were evoked by the bath application of acetylcholine (1 μ M or higher). The rise in [Ca 2+] i was due to the release of Ca 2+ from intracellular Ca 2+ stores, since the Ca 2+ response occurred even in a Ca 2+-free medium. The Ca 2+ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μ M abolisehd the response to 10 μ M acetylcholine; muscarine and carbamylcholine (100 μ M each) evoked Ca 2+ rises. Increases in [Ca 2+] i were also evoked by the bath application of ATP (10 μ M or higher). The Ca 2+ rise by ATP was evoked even in a Ca 2+-free medium. Adenosine (500 μ M) did not cause any Ca 2+ response. Suramin and reactive blue 2 (200 μ M each) completely blocked the Ca 2+ response to 500μ M ATP. Uridine triphosphate (500 μ M) caused comparable Ca 2+ responses with those to 500 μ M ATP. These results suggested the involvement of P 2U purinoceptors. The potentiation of Ca 2+ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μ M and 100 μ M, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa 2+ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc. 相似文献
16.
The effect of carvedilol on cytosolic free Ca 2+ concentrations ([Ca 2+] i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca 2+] i levels in suspended OC2 cells by using fura-2 as a Ca 2+-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca 2+] i in a concentration-dependent fashion. The Ca 2+ signal was decreased by 50% by removing extracellular Ca 2+. Carvedilol-induced Ca 2+ entry was not affected by the store-operated Ca 2+ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca 2+-free medium, incubation with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin did not change carvedilol-induced [Ca 2+] i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca 2+ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca 2+] i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca 2+] i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca 2+ was chelated with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca 2+] i rise by causing phospholipase C-independent Ca 2+ release from mitochondria and non-endoplasmic reticulum stores, and Ca 2+ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca 2+-independent manner that involved apoptosis. 相似文献
17.
Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca 2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca 2+ sources: (a) Ca 2+ surrounding single cells was transiently displaced by applying agonist with or without Ca 2+ from an ejection pipette. (b) Intracellular stores of Ca 2+ were depleted by soaking cells in Ca 2+-free plus 1 m M EGTA solution before transient exposure to agonist plus Ca 2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca 2+ concentration ([Ca 2+] i) was measured by indo-1 fluorescence. KCl (35 m M) and nicotine (10 µ M) caused an immediate increase in [Ca 2+] i and secretion in cells with or without internal Ca 2+ stores, but only when applied with Ca 2+ in the ejection pipette. Caffeine (10 m M) and muscarine (30 µ M) evoked exocytosis whether or not Ca 2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca 2+ stores. Pretreatment with ryanodine (0.1 µ M) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca 2+] i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ~20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca 2+] i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca 2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca 2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland. 相似文献
18.
The effect of extracellular calcium ([Ca 2+]
e
) on cytosolic calcium ([Ca 2+]
i
) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In
cortical collecting ducts, basolateral but not apical changes in [Ca 2+]
e
were associated with parallel changes in [Ca 2+]
i
. Basal [Ca 2+]
i
was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La 3+. Increasing peritubular [Ca 2+]
e
triggered Ca 2+ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca 2+-receptor RaKCaR, e.g., Ba 2+, Mg 2+, Gd 3+, and neomycin, but was reproduced by Ni 2+. Ni 2+-induced mobilization of intracellular Ca 2+ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca 2+]
e
.
In the cortical thick ascending limb, removing basolateral Ca 2+ hardly altered [Ca 2+]
i
but increasing [Ca 2+]
e
or adding Ba 2+, Mg 2+, Gd 3+ and neomycin released intracellular calcium.
These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions;
(2) this influx occurs through nonvoltage gated channels, permeable to Ba 2+, insensitive to verapamil and nifedipine, and blocked by La 3+; (3) increasing [Ca 2+]
e
stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR;
(4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni 2+ but not to Ca 2+ is present in the collecting duct.
Received: 12 July 1996/Revised: 28 October 1996 相似文献
19.
A partially purified preparation of the lobster muscle Na +/Ca 2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca 2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular
( ev) medium. Extravesicular Na + (Na
ev
)-dependent Ca 2+ efflux depended directly upon the extravesicular Ca 2+ concentration ([Ca 2+]
ev
) with a half-maximal activation at [Ca 2+]
ev
= 0.6 μ m. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca 2+, as in most other species. In contrast, at low [Na +]
ev
, the Ca
ev
-binding site (i.e., on the cytoplasmic surface) for Ca 2+ transported via Ca 2+/Ca 2+ exchange was half-maximally activated by about 7.5 μ m Ca 2+. Mild proteolysis of the Na +/Ca 2+ exchanger by α-chymotrypsin also upregulated the Na
ev
-dependent Ca 2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca 2+. Proteolytic digestion in the presence of 1.9 μ m free ev Ca 2+, however, induced only a 1.6-fold augmentation of Ca 2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca 2+ also inhibited proteolytic degradation of the Na +/Ca 2+ exchanger measured by immunoblotting. These data suggest that Ca 2+, bound to a high affinity binding site, protects against the activation of the Na +/Ca 2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na +/Ca 2+ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 m m, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster
body fluids.
Received: 20 October 1999/Revised: 13 January 2000 相似文献
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